Oxidative reactions peroxidases

Detecting the presence of small, even invisible, amounts of blood is routine. Physical characteristics of dried stains give minimal information, however, as dried blood can take on many hues. Many of the chemical tests for the presence of blood rely on the catalytic peroxidase activity of heme (56,57). Minute quantities of blood catalyze oxidation reactions between colorless materials, eg, phenolphthalein, luco malachite green, luminol, etc, to colored or luminescent ones. The oxidant is typically hydrogen peroxide or sodium perborate (see Automated instrumentation,hematology). 

The enzymatic reactions of peroxidases and oxygenases involve a two-electron oxidation of iron(III) and the formation of highly reactive [Fe O] " species with a formal oxidation state of +V. Direct (spectroscopic) evidence of the formation of a genuine iron(V) compound is elusive because of the short life times of the reactive intermediates [173, 174]. These species have been safely inferred from enzymatic considerations as the active oxidants for several oxidation reactions catalyzed by nonheme iron centers with innocent, that is, redox-inactive, ligands [175]. This conclusion is different from those known for heme peroxidases and oxygenases... 

Takahama, U. Oxidation of hydroxycinnamic acid and hydroxycinnamyl alcohol derivatives by laccase and peroxidase—interactions among p-hydroxyphenyl, guaiacyl and syringyl groups during the oxidation reactions. Physiol. Plantarum 1995, 93, 61-68. 

Several compounds can be oxidized by peroxidases by a free radical mechanism. Among various substrates of peroxidases, L-tyrosine attracts a great interest as an important phenolic compound containing at 100 200 pmol 1 1 in plasma and cells, which can be involved in lipid and protein oxidation. In 1980, Ralston and Dunford [187] have shown that HRP Compound II oxidizes L-tyrosine and 3,5-diiodo-L-tyrosine with pH-dependent reaction rates. Ohtaki et al. [188] measured the rate constants for the reactions of hog thyroid peroxidase Compounds I and II with L-tyrosine (Table 22.1) and showed that Compound I was reduced directly to ferric enzyme. Thus, in this case the reaction of Compound I with L-tyrosine proceeds by two-electron mechanism. In subsequent work these authors have shown [189] that at physiological pH TPO catalyzed the two-electron oxidation not only L-tyrosine but also D-tyrosine, A -acetyltyrosinamide, and monoiodotyrosine, whereas diiodotyrosine was oxidized by a one-electron mechanism. 

Various hydroxyl and amino derivatives of aromatic compounds are oxidized by peroxidases in the presence of hydrogen peroxide, yielding neutral or cation free radicals. Thus the phenacetin metabolites p-phenetidine (4-ethoxyaniline) and acetaminophen (TV-acetyl-p-aminophenol) were oxidized by LPO or HRP into the 4-ethoxyaniline cation radical and neutral V-acetyl-4-aminophenoxyl radical, respectively [198,199]. In both cases free radicals were detected by using fast-flow ESR spectroscopy. Catechols, Dopa methyl ester (dihydrox-yphenylalanine methyl ester), and 6-hydroxy-Dopa (trihydroxyphenylalanine) were oxidized by LPO mainly to o-semiquinone free radicals [200]. Another catechol derivative adrenaline (epinephrine) was oxidized into adrenochrome in the reaction catalyzed by HRP [201], This reaction can proceed in the absence of hydrogen peroxide and accompanied by oxygen consumption. It was proposed that the oxidation of adrenaline was mediated by superoxide. HRP and LPO catalyzed the oxidation of Trolox C (an analog of a-tocopherol) into phenoxyl radical [202]. The formation of phenoxyl radicals was monitored by ESR spectroscopy, and the rate constants for the reaction of Compounds II with Trolox C were determined (Table 22.1). 

He got a Habilitation a diriger les recherches in 2008 and he is now developing his own project that consists of the elaboration of new hybrid metalloprotein catalysts for selective oxidation reactions, by insertion of metal cofactors into xylanases. He then studies their peroxidase, catalase, and monooxygenase activities, in particular in the selective oxidation of sulfides, alkanes, and alkenes. 

Oxidation of organic contaminants by microorganisms is one of the basic metabolic reactions in the subsurface and involves the presence of a group of oxidative enzymes such as peroxidases, lactases, and mixed-function oxidizes. Major oxidative reactions that may occur in the subsurface are presented and explained in Table 15.2. 

The oxidation reactions of luminol and lucigenin can be used to assay for H Oj. For example, analysis of glucose in biological systems can be achieved using a three-enzyme system of mutarotase, glucose oxidase and horseradish peroxidase by correlation with the amount of HjOj released. Similarly, cholesterol can be measured using cholesterol oxidase. The fact that the rate of luminol oxidation depends on the concentration of the catalyst can be used as a method for determination of Co +, Fe +, Cr + and Mn + and other catalysts.Some examples of the use of luminol, isolumi-nol and their derivatives in immunoassays are shown in Table 3.11. ... 

A comparison of peroxidase and cytochrome P-450 illustrates the problems of comparing enzymes and their related catalysts such as synzymes. Peroxidase has low substrate specificity and a simple free-radical oxidation reaction. The substrate site is 10 A from the iron (H202 site) and is probably just an oily droplet region of the protein. This proteins has parallels with Professor Klotz s systems. Proximity is perhaps sufficient to explain the activation of the organic substrate (but not for that of H2Oz). 

For foreign compounds, the majority of oxidation reactions are catalyzed by monooxygenase enzymes, which are part of the mixed function oxidase (MFO) system and are found in the SER (and also known as microsomal enzymes). Other enzymes involved in the oxidation of xenobiotics are found in other organelles such as the mitochondria and the cytosol. Thus, amine oxidases located in the mitochondria, xanthine oxidase, alcohol dehydrogenase in the cytosol, the prostaglandin synthetase system, and various other peroxidases may all be involved in the oxidation of foreign compounds. 

Other than for the monooxygenases, a two-electron acceptor such as hydrogen peroxide is required as the terminal oxidant for peroxidases. In the so-called resting state the Fe ion is situated in the oxidation state +3. Reaction with hydrogen peroxide proceeds with loss of water and yields a ferryl(IV) radical cation called Compound I,... 

Preparations of HRP may vary in their carbohydrate content and this can affect the oxidation reaction. Free carbohydrate can be removed by gel filtration. Increasing the sodium periodate concentration to 0.2 M can also help, but further increases lead to inactivation of the peroxidase. 

In the context of the above, the authors suggest that consideration of catalase and non-classical peroxidase reactions from positions of the ability of H202 to induce chemical conjugation in oxidation reactions broadens our knowledge about the role and mechanism of catalases and peroxidases. 

Critically analyzing the mechanism (6.8)-(6.12), one may note the unsuitability of the currently presented interaction between complexes E-Fe3+—OH and E-Fe3+ OOH and substrates (H202 and H2D), because it is unclear how the substrate is activated. Moreover, intensification of the catalase reaction induces a non-classical peroxidase activity increase in ethanol and formic acid oxidation reactions. This indicates the existence of a unit common to these two processes [82, 83], The alternative action of catalase (catalase of peroxidase reaction) in the biosystem with solidarity of elementary stage mechanisms should be noted [88, 89], Peroxidase action of catalase requires a continuous supply of H202 for ethanol and formic acid oxidation, which can be explained by oxidation according to conjugated mechanism [90],... 

At least two different catalytic sites have been suggested for the one- and two-electron oxidative reactions catalyzed by CPO [89]. Phenols and large substrates unable to gain access to the heme are presumably oxidized at the enzyme surface by (one-electron) LRET pathways similar to that described in Sect. 3.3.3 for lignino-lytic peroxidases, although the catalytic residue exposed to the solvent has not been yet identified. 

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